Lyme disease is the most common tick-borne illness in the United States and Europe. It is caused by Borrelia burgdorferi, a bacterial pathogen that is maintained in nature in a zoonotic cycle between various species of small mammals and an ixodid tick vector. A hallmark of the Lyme disease spirochete is its unusual segmented genome, which includes a large number of linear and circular plasmids. Increasing evidence indicates that plasmid-encoded genes are critical for successful adaptation to the different environments that B. burgdorferi encounters during its infectious cycle. We have developed genetic tools to investigate basic aspects of the unusual genomic organization, cellular structure and metabolism of B. burgdorferi. We have extended this investigation to an in vivo setting with an experimental system that closely mimics the natural arthropod vector/rodent host infectious cycle. Through an understanding of the basic molecular biology of the organism, we hope to gain insight into the infectious strategy utilized by this significant vector-borne pathogen and thereby facilitate efforts to prevent, diagnose and treat Lyme disease. B. burgdorferi displays different proteins on its surface at different stages of the infectious cycle, a phenotype that we have termed lipoprotein succession. These prominent and abundant surface components are plasmid-encoded and tethered to the bacterial outer membrane by an amino-terminal acyl group, which forms the basis for their designation as lipoproteins. Outer surface protein A (OspA) coats the spirochete when it is acquired from an infected host by a feeding tick and facilitates spirochete colonization of the tick midgut. An unrelated lipoprotein, OspC, appears on the surface of B. burgdorferi several months later, when the tick feeds again and spirochetes are transmitted to a new host. We previously demonstrated that OspC is an essential virulence factor that B. burgdorferi requires at the initial stage of mammalian infection. Finally, a third plasmid-encoded lipoprotein, VlsE, replaces OspC on the surface of the spirochete after infection is established; VlsE undergoes antigenic variation and allows the spirochete to evade host acquired immunity. Wild type B. burgdorferi can persist indefinitely in the tick vector and vertebrate host, growing exponentially when nutrients are available and assuming a metabolically inert state when nutrients are limiting. In FY2016 we published results from our ongoing investigation of lipoprotein succession and other adaptive mechanisms employed by B. burgdorferi during the successful completion of its infectious cycle (Tilly et al., ref #1). Dr. Kit Tilly, a senior research assistant, constructed a B. burgdorferi variant (ApC) in which a second copy of the ospC gene was placed under the control of the ospA promoter at the ospA/B locus on its native plasmid; these spirochetes made OspC instead of OspA when entering and colonizing the tick midgut. The endogenous ospC locus on a different native plasmid remained intact, thus allowing ApC spirochetes to continue to synthesize OspC from this gene copy during tick feeding and transmission to the mammalian host. When assessed in the experimental mouse-tick infectious cycle, ApC spirochetes were fully proficient for host infection, but highly attenuated for tick colonization. This tick colonization defect was less severe when ApC spirochetes were acquired from infected SCID mice, which lack acquired immunity. These data extend previous studies indicating that OspA shelters spirochetes in the tick midgut from antibodies in the ingested blood meal, while providing an additional function during tick colonization. This finding confirms our previous conclusion that OspC and OspA fulfill non-overlapping, distinct and critical functions in the vertebrate host and arthropod vector, respectively. Dr. Tilly retired at the end of 2015; we remain grateful and indebted to Kit for her significant contributions to this project and the Molecular Genetics Section throughout the years. In FY2016 we published an assessment of BB0449, a borrelia homolog of a protein termed hibernation-promoting factor (HPF), during spirochete survival under prolonged nutrient-limited conditions (Fazzino et al., ref #2). This study was conducted by a post-baccalaureate IRTA trainee, Lisa Fazzino, who worked closely with Dr. Kit Tilly. In other bacteria, HPF has been shown to dimerize ribosomes and prevent translation of mRNA, thereby contributing to bacterial survival during stationary phase. By secondary structure modeling, BB0449 was predicted to resemble HPF, but BB0449 did not co-localize with ribosomes during cellular fractionation of B. burgdorferi, and protein and transcript levels remained low and unmodulated at all phases of growth. In addition, B. burgdorferi mutants lacking BB0449 exhibited a wild-type phenotype throughout the experimental infectious cycle. We conclude that BB0449 in B. burgdorferi does not participate in growth cessation through binding ribosomes during prolonged stationary phase survival in unfed ticks or at any other stage of infection. Ms. Fazzino is currently a graduate student in a doctoral program at the University of Minnesota, Minneapolis/St. Paul. In FY2016 we completed and published a study on homologs of the FtsH protease and its modulators HflK and HflC, in B. burgdorferi (Chu, Stewart et al., ref. #3). This study was conducted by a Visiting Fellow, Dr. Chenyi Chu, under the supervision of Dr. Philip Stewart, a Staff Scientist in the MGS. In other bacteria, FtsH, HflK and HflC form a large complex that participates in membrane quality control and cellular response to stress, which are critical for membrane structure and function. After repeated unsuccessful attempts to inactivate FtsH in B. burgdorferi, Drs. Chu and Stewart used an inducible system to demonstrate that FtsH provides an essential function for the Lyme disease spirochete. No permissive growth conditions were found in which spirochetes could survive without FtsH. In contrast, HflK and HflC were dispensable for B. burgdorferi growth both in vitro and in vivo, and no restrictive conditions were identified in which mutants lacking these putative FtsH modulators were distinguishable from wild type. We conclude that HflK and HflC are not required for FtsH essential function. Dr. Chu is currently an assistant researcher at the Beijing Institute of Disease Control and Prevention, P.R. China. In FY 2016 we published the results of a study comparing the virulence of B. burgdorferi in ticks before and after feeding on a host (Kasumba et al., ref. #4). This study was initiated by a former Visiting Fellow, Dr. Irene Kasumba. She found that B. burgdorferi derived from unfed ticks were viable but essentially non-pathogenic, even with an inoculum of 10,000 organisms, while as few as 10 spirochetes from fed ticks could reliably cause infection. Engineered production of the virulence factor OspC by B. burgdorferi in unfed ticks did not over-ride their non-infectious phenotype. These results demonstrate that conditional priming of B. burgdorferi during tick feeding is critical for spirochete infectivity and indicate that virulence factors additional to OspC comprise this adaptive response. Investigation of these unidentified yet critical factors will provide a better understanding of the basic biology of the Lyme disease spirochete and should form a basis for the rational design of new ways to prevent and treat human infection by this tick-borne pathogen. Dr. Kasumba is currently a research assistant at the Center for Vaccine Development in the Institute of Global Health at the University of Maryland, Baltimore.